The traditional methods for gene discovery, including chemical mutagenesis, irradiation and T-DNA insertion, used to screen loss of function mutants have limitations. Mutagenic methods such as these rarely identify genes that are redundant in the genome, and gene characterization is time-consuming and laborious.
Activation tagging is a method by which genes are randomly and strongly up-regulated on a genome-wide scale, after which specific phenotypes are screened for and selected. Isolation of mutants by activation tagging has been reported (Hayashi et al., 1992). An activation T-DNA tagging construct was used to activate genes in tobacco cell culture allowing the cells to grow in the absence of plant growth hormones (Walden et al., 1994). Genes have been isolated from plant genomic sequences flanking the T-DNA tag and putatively assigned to plant growth hormone responses. (See, e.g., Miklashevichs et al. 1997, Harling et al., 1997; Walden et. al., 1994; and Schell et al., 1998, which discusses related studies.)
The first gene characterized in Arabidopsis using activation tagging was a gene encoding the histone kinase involved in the cytokinin signal transduction pathway. The gene sequence was isolated from plant genomic DNA by plasmid rescue and the role of the gene, CKI1, in cytokinin responses in plants was confirmed by re-introduction into Arabidopsis (Kakimoto, 1996). This was followed by reports of several dominant mutants such as TINY, LHY and SHI using a similar approach along with the Ds transposable element (Wilson et al., 1996, Schaffer et al., 1998, Fridborg et al., 1999). In a more recent report, activation T-DNA tagging and screening plants for an early flowering phenotype led to the isolation of the FT gene (Kardailsky et al., 1999).
The potential application of activation tagging as a high through put technology for gene discovery has been demonstrated based on screening of several dominant mutant genes involved in photoreceptor, brassinosteroid, gibberellin and flowering signal pathways, as well as disease resistance. (See, e.g., Weigel et al., 2000, Christensen et al., 1998; Kardailsky et al., 1999).
Arabidopsis has been widely used as a model for plant improvement for plants such as Brassica species having a siliques type of fruit. However, Arabidopsis does not serve as a model for plants having a fleshy fruit.
A method for identifying and characterizing genes based on modified gene expression in fruit-bearing plants is described in PCT publication WO0053794. Dwarf varieties of fruit-bearing plants, particularly dwarf varieties of tomato, are useful in the overexpression of one or more native plant genes and in correlating that overexpression with a particular phenotype.
Dwarf tomatoes are characterized by their short intemodes, which give plants a compact appearance. The miniature Lycopersicon esculentum cultivar, Micro-Tom is a proportionally dwarfed plant that grows at high density (up to 1357 plants/m−2), has a short life cycle (70–80 days from sowing to fruit ripening), and for which fruit size, and leaf size have been genetically reduced (Meissner et al., 1997; Scott and Harbaugh, 1989). In addition, Micro-Tom has been shown to be resistant to a number of diseases and can be transformed at frequencies of up to 80% through Agrobacterium-mediated transformation of cotyledons (Meissner et al., 1997). Similar to Micro-Tom, Florida Petite (Fla. Agr. Expt. Sta. Circ. S-285), Tiny Tim and Small Fry are dwarf varieties of tomato which have a short life cycle, and for which fruit size, and leaf size have been genetically reduced.
Efforts are underway in industry and academia to develop a means to identify genes associated with particular plant traits or characteristics in order to develop improved plants having such traits. The present invention provides a plant phenotype associated with modified expression of a native plant gene.
In an activation tagging screen in Micro-Tom, we identified a gene involved in pigment production. Anthocyanins are pigments that are responsible for many of the red and blue colors in plants. The genetic basis of anthocyanin biosynthesis has been well characterized in corn, Petunia, and Antirrhinium (Dooner et al, 1991; Jayaram and Peterson, 1990; Quattrocchio F et al., 1999).